Modification of PCR conditions and design of exon-specific primers for the efficient molecular diagnosis of PKD1 mutations.

BACKGROUND/AIMS Autosomal-dominant polycystic kidney disease (ADPKD) is a heterogeneous genetic disorder caused by mutations in the PKD1 and PKD2 genes. Currently, long-range PCR followed by nested PCR and sequencing (LRNS) is the gold standard approach for PKD1 testing. However, LRNS is complicated by the high structural and sequence complexity of PKD1, which makes the procedure for amplification and analysis of PKD1 difficult. METHODS Here in, we modified the PCR conditions and designed primers for efficient and specific amplification of both the long-range and individual exons of PKD1. RESULTS Using the modified system, seven long-range fragments were specifically amplified using two distinct sets of conditions, and all individual exon PCR assays were easily performed using a touch-down PCR method. Seven pathogenic or likely pathogenic variants, including two novel truncated frameshift indels and two novel likely pathogenic missense mutations, were identified in eight unrelated patients with or without histories of ADPKD disease (one variant was observed in two unrelated patients). Using combined bioinformatics tools, two indeterminate missense variants were identified in two sporadic patients. CONCLUSION Four novel PKD1 variants were identified in this study. We demonstrated that the modified LRNS method achieves high sensitivity and specificity for detecting pathogenic variants of ADPKD.